Development of Delivery Systems Enhances the Potency of Cell-Based HIV-1 Therapeutic Vaccine Candidates.

An effective therapeutic vaccine to eradicate HIV-1 infection does not exist yet. Among different vaccination strategies, cell-based vaccines could achieve in clinical trials. Cell viability and low nucleic acid expression are the problems related to dendritic cells (DCs) and mesenchymal stem cells (MSCs), which are transfected with plasmid DNA. Thus, novel in vitro strategies are needed to improve DNA transfection into these cells. The recent study assessed immune responses generated by MSCs and DCs, which were derived from mouse bone marrow and modified with Nef antigen using novel methods in mice. For this purpose, an excellent gene transfection approach by mechanical methods was used. Our data revealed that the transfection efficacy of Nef DNA into the immature MSCs and DCs was improved by the combination of chemical and mechanical (causing equiaxial cyclic stretch) approaches. Also, chemical transfection performed two times with 48-hour intervals further increased gene expression in both cells. The groups immunized with Nef DC prime/rNef protein boost and then Nef MSC prime/rNef protein boost were able to stimulate high levels of IFN-γ, IgG2b, IgG2a, and Granzyme B directed toward Th1 responses in mice. Furthermore, the mesenchymal or dendritic cell-based immunizations were more effective compared to protein immunization for enhancement of the Nef-specific T-cell responses in mice. Hence, the use of chemical reagent and mechanical loading simultaneously can be an excellent method in delivering cargoes into DCs and MSCs. Moreover, DC- and MSC-based immunizations can be considered as promising approaches for protection against HIV-1 infections.

HIV-1 Nef is released in extracellular vesicles derived from astrocytes: evidence for Nef-mediated neurotoxicity.

Human immunodeficiency virus-associated neurological disorders (HANDs) affect the majority of AIDS patients and are a significant problem among HIV-1-infected individuals who live longer because of combined anti-retroviral therapies. HIV-1 utilizes a number of viral proteins and subsequent cytokine inductions to unleash its toxicity on neurons. Among HIV-1 viral proteins, Nef is a small protein expressed abundantly in astrocytes of HIV-1-infected brains and has been suggested to have a role in the pathogenesis of HAND. In order to explore its effect in the central nervous system, HIV-1 Nef was expressed in primary human fetal astrocytes (PHFAs) using an adenovirus. Our results revealed that HIV-1 Nef is released in extracellular vesicles (EVs) derived from PHFA cells expressing the protein. Interestingly, HIV-1 Nef release in EVs was enriched significantly when the cells were treated with autophagy activators perifosine, tomaxifen, MG-132, and autophagy inhibitors LY294002 and wortmannin suggesting a novel role of autophagy signaling in HIV-1 Nef release from astrocytes. Next, Nef-carrying EVs were purified from astrocyte cultures and neurotoxic effects on neurons were analyzed. We observed that HIV-1 Nef-containing EVs were readily taken up by neurons as demonstrated by immunocytochemistry and immunoblotting. Furthermore, treatment of neurons with Nef-carrying EVs induced oxidative stress as evidenced by a decrease in glutathione levels. To further investigate its neurotoxic effects, we expressed HIV-1 Nef in primary neurons by adenoviral transduction. Intracellular expression of HIV-1 Nef caused axonal and neurite degeneration of neurons. Furthermore, expression of HIV-1 Nef decreased the levels of phospho-tau while enhancing total tau in primary neurons. In addition, treatment of primary neurons with Nef-carrying EVs suppressed functional neuronal action potential assessed by multielectrode array studies. Collectively, these data suggested that HIV-1 Nef can be a formidable contributor to neurotoxicity along with other factors, which leads to HAND in HIV-1-infected AIDS patients.

Naloxone/alum mixture a potent adjuvant for HIV-1 vaccine: induction of cellular and poly-isotypic humoral immune responses.

In the present study we used a fusion peptide from HIV-1 p24 and Nef as vaccine model and adjuvant activity of Naloxone/alum mixture was evaluated in a peptide vaccine model. HIV-1 p24-Nef fusion peptide was synthesized. Female BALB/c mice were divided into five groups. The first group immunized subcutaneously with the p24-Nef fusion peptide adjuvanted with Naloxone/alum mixture and boosted with same protocol. The second was immunized with fusion peptide adjuvanted in alum. The control groups were injected with NLX (Group 3), Alum (Group 4), or PBS (Groups 5) under the same conditions. To determine the type of induced immune response, sera and splenocytes were analyzed by commercial ELISA method for total IgG and isotypes and cytokine secretion (IL-4 & IFN-γ), respectively. We have also used the ELISPOT assay to monitor changes in the frequency of IFN-γ-producing T cells. The proliferation of T cells was assessed using Brdu method and T-cell cytotoxicity was assessed with CFSE method. Immunization of mice with HIV-1 p24-Nef fusion peptide formulated in Naloxone/alum mixture significantly increased lymphocyte proliferation and shifted cytokine responses toward Th1 profile compared to all other groups. Analysis of humoral immune responses revealed that administration of HIV-1 p24-Nef fusion peptide with Naloxone/alum mixture significantly increased specific IgG responses and also increased IgG1,IgG2a, IgG2b, IgG3, and IgM vs. alum-adjuvanted vaccine groups. Naloxone/alum mixture as an adjuvant could improve cellular and humoral immune response for HIV vaccine model and this adjuvant maybe useful for HIV vaccine model in human clinical trial.

Short conserved sequences of HIV-1 are highly immunogenic and shift immunodominance.

UNLABELLED: Cellular immunity is pivotal in HIV-1 pathogenesis but is hampered by viral sequence diversity. An approach to minimize this diversity is to focus immunity on conserved proteome sequences; therefore, we selected four relatively conserved regions (Gag amino acids 148 to 214 and 250 to 335, Env amino acids 521 to 606, and Nef amino acids 106 to 148), each created in three mosaics, to provide better coverage of M-group HIV-1 sequences. A conserved-region vaccine (CRV) delivering genes for these four regions as equal mixtures of three mosaics each (each region at a separate injection site) was compared to a whole-protein vaccine (WPV) delivering equimolar amounts of genes for whole Gag, Env, and Nef as clade B consensus sequences (separate injection sites). Three rhesus macaques were vaccinated via three DNA primes and a recombinant adenovirus type 5 boost (weeks 0, 4, 8, and 24, respectively). Although CRV inserts were about one-fifth that of WPV, the CRV generated comparable-magnitude blood CD4+ and CD8+ T lymphocyte responses against Gag, Env, and Nef. WPV responses preferentially targeted proteome areas outside the selected conserved regions in direct proportion to sequence lengths, indicating similar immunogenicities for the conserved regions and the outside regions. The CRV yielded a conserved-region targeting density that was approximately 5-fold higher than that of the WPV. A similar pattern was seen for bronchoalveolar lymphocytes, but with quadruple the magnitudes seen in blood. Overall, these findings demonstrate that the selected conserved regions are highly immunogenic and that anatomically isolated vaccinations with these regions focus immunodominance compared to the case for full-length protein vaccination. IMPORTANCE: HIV-1 sequence diversity is a major barrier limiting the capability of cellular immunity to contain infection and the ability of vaccines to match circulating viral sequences. To date, vaccines tested in humans have delivered whole proteins or genes for whole proteins, and it is unclear whether including only conserved sequences would yield sufficient cellular immunogenicity. We tested a vaccine delivering genes for four small conserved HIV-1 regions compared to a control vaccine with genes for whole Gag, Env, and Nef. Although the conserved regions ranged from 43 to 86 amino acids and comprised less than one-fifth of the whole Gag/Env/Nef sequence, the vaccines elicited equivalent total magnitudes of both CD4+ and CD8+ T lymphocyte responses. These data demonstrate the immunogenicity of these small conserved regions and the potential for a vaccine to steer immunodominance toward conserved epitopes.

Immunogenicity of chloroplast-derived HIV-1 p24 and a p24-Nef fusion protein following subcutaneous and oral administration in mice.

High-level expression of foreign proteins in chloroplasts of transplastomic plants provides excellent opportunities for the development of oral vaccines against a range of debilitating or fatal diseases. The HIV-1 capsid protein p24 and a fusion of p24 with the negative regulatory protein Nef (p24-Nef) accumulate to ∼4% and ∼40% of the total soluble protein of leaves of transplastomic tobacco (Nicotiana tabacum L.) plants. This study has investigated the immunogenicity in mice of these two HIV-1 proteins, using cholera toxin B subunit as an adjuvant. Subcutaneous immunization with purified chloroplast-derived p24 elicited a strong antigen-specific serum IgG response, comparable to that produced by Escherichia coli-derived p24. Oral administration of a partially purified preparation of chloroplast-derived p24-Nef fusion protein, used as a booster after subcutaneous injection with either p24 or Nef, also elicited strong antigen-specific serum IgG responses. Both IgG1 and IgG2a subtypes, associated with cell-mediated Th1 and humoral Th2 responses, respectively, were found in sera after subcutaneous and oral administration. These results indicate that chloroplast-derived HIV-1 p24-Nef is a promising candidate as a component of a subunit vaccine delivered by oral boosting, after subcutaneous priming by injection of p24 and/or Nef.